Title: Cloning, expression, and purification of a sesquiterpene cyclase from geosmin-producing Oscillatoria splendida Authors
Submitted to: International Journal of Applied Agricultural Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 26, 2012
Publication Date: January 10, 2013
Citation: Hurlburt, B.K., Fisher, A.L., Mcbride, J.K., Noss, K. 2013. Cloning, expression, and purification of a sesquiterpene cyclase from geosmin-producing Oscillatoria splendida. International Journal of Applied Agricultural Research. 8:1-11. Interpretive Summary: Aquaculture is an important growing part of agriculture, mainly due to the depletion of natural fisheries. The majority of aquaculture systems are closed, meaning there is not a constant influx of fresh water. As a result of being closed and having large amounts of nutrient rich feed introduced, micro-organisms are abundant. Cyanobacteria are commonly found in these systems, and some of them produce off-flavor compounds that make the fish unmarketable. Our goal is to develop methods of inhibiting the accumulation of these compounds. Towards that goal, we have cloned the gene for an important enzyme involved in the final step of the synthesis of geosmin, one of two main off-flavor compounds. We have expressed and purified recombinant protein that can be used to develop inhibitors that may be used in aquaculture systems.
Technical Abstract: The aquaculture industry is a growing part of agriculture worldwide. Many of these systems, primarily closed ones, encounter serious issues with off-flavors. Cyanobacteria are the main producers of the two dominant off-flavor compounds, geosmin and 2-methylisoborneol. In an effort to begin to understand the biosynthesis of these compounds, we have cloned a gene homologous to ones that are involved in geosmin biosynthesis in other organisms. The encoded protein was expressed using a T7 system in E. coli. Initially, the majority of the expressed protein was insoluble. Here we report conditions for expression of soluble protein, and the denaturation/renaturation of insoluble protein. Having this enzyme will allow the characterization of its activity and testing compounds that may be used to inhibit it.